CFTR is a member of the ABC Transporter superfamily, but is the only member known to bear ion channel activity. Using a molecular evolution analysis, we have identified residues in CFTR that appear to be critical to the evolutionary transition from transporter to channel. The long-term objective is to understand how the pore of the CFTR channel changes its structure between the open and closed states, how steps in the ATP-dependent gating cycle control pore gating, and how CFTR evolved the capability to interrupt a transporter mechanism in order to gain ion channel function. This proposal will test the hypothesis that chloride channel activity evolved in CFTR by converting the conformational changes in the membrane domain associated with binding and hydrolysis of ATP at the cytoplasmic domains, as are found in true ABC Transporters, into the formation of a stable open state, by means of inter- and intra-domain interactions. Results from the previous funding period show that disruption of intradomain interactions in CFTR, at sites that are also identified as divergent between CFTR and related ABC Transporters, dramatically alter channel behavior in terms of conductance, selectivity, pharmacology, and transitions between multiple conducting states. This renewal application proposes to use a refined evolutionary analysis, coupled to structure/function experiments based upon quantitative electrophysiological assays of CFTR channels expressed in oocytes, and simulations of CFTR homology models, to test the importance of specific residues in CFTR in the switch from transporter to channel. Aim #1 is to identify residues that underlie the evolution of channel activity in CFTR. At sites that exhibit evolutionary divergence from transporters, the impact of mutations on channel activity will be determined. Aim #2 is to determine how ATP-dependent gating at the cytoplasmic domains leads to conformational changes in the pore associated with channel function. This Aim will include both experiment and molecular simulation. At sites predicted to interact to stabilize each of the open conductance states, the rate and state-dependence of crosslinking with bifunctional sulfhydryl-modifying (SH) reagents of various lengths will be determined, allowing us to construct and test a scheme for the movements in the pore associated with both opening and closing. These results will be used to validate molecular dynamics simulations of selected CFTR homology models, to identify which structures most closely reflect the true channel structure. Aim #3 is to verify the importance of the interacting residues by assessing their role in transporter function, in both CFTR and a related glutathione transporter. It is expected that interactions that promote channel behavior will disrupt transporter behavior, which may impact drug development for CF. These studies will allow us to associate molecular motions in CFTR's pore domain with steps in the ATP- dependent gating cycle, will allow us to identify residues that are critical to ion channel function in CFTR, and may identify sites where drugs can be docked to lock open CFTR channels, leading to increased Cl- secretion.